整合HA蛋白的HIV假病毒展示禽流感病毒感染宿主细胞机制

通过将高致病性禽流感病毒HA蛋白整合到HIV颗粒，包装成表达HA蛋白的假病毒粒子（命名为HIV/H5-HA），并对所包装的假病毒的生物学功能进行了研究.通过RT PCR获得了H5N1亚型禽流感病毒完整的血凝素基因(HA)并克隆到真核表达载体pcDNA3.1(+)上，通过与假病毒构建体系的2种质粒pCMV△8.2和pHR′-CMVLacZ共转染293T细胞,包装成假病毒颗粒.利用LacZ染色和HA假病毒颗粒感染MDCK等6种细胞株并对标记基因LacZ进行检测.结果表明,HIV/H5-HA与天然的禽流感病毒相似，具有广泛的细胞嗜性； Western 印迹和FACS检测结果,和HA假病毒颗粒的电镜照片确认了HA基因在假病毒颗粒表面得到了表达；HIV/H5-HA能够凝集鸡红细胞，并且pH值依赖性测定表明,HA假病毒需要低pH值才能实现正确的入侵宿主细胞.本研究结果显示：禽流感病毒H5N1亚型的HA基因得到了有效的包装，并且所包装的假病毒颗粒能够表达具有高度生物活性的HA蛋白.同时，假病毒模型的建立为进一步研究禽流感病毒与宿主之间的免疫应答提供了一种新的途径.

Modified Cell Entry by Exhibiting of Hemagglutinin from Avian Influenza Virus on Pseudotyped HIV Particles

The pseudotyped virus (HIV/H5 HA) was produced into HIV particles by incorporating hemagglutinin (HA) protein from H5N1 avian influenza virus (AIV) and its infectivity was characterized in various cell lines. The HA fragment was amplified by RT- PCR and cloned into a pcDNA3.1(+) vector, then cotransfected with retroviral producing vectors pCMV△8.2 and pHR′-CMVLacZ into 293T cells for viral packaging. The HIV/H5-HA transduction was analysis by X-Gal Staining after infections to 293T, BHK, Vero, PK-15, IBRS-2 and MDCK cells. The results demonstrated that the pseudotyped viral particles possessed a wide cell tropism. The HA glycoproteins were properly decorated on the virions as determined by Western blot, FACS and EM, and were able to agglutinate chicken erythrocytes. We also found that HIV/H5-HA entry into the infected cells required a low pH for membrane fusions. Our results showed that the HA-presenting HIV pseudotyping system was capable to produce functional virions, and might be a useful for the study of the host immunologic response of AIV.